Abstract Rhinovirus (RV) is the most common cause of asthma exacerbation but underlying mechanisms are not com- pletely known. We have noted colocalization of RV with CD68, a macrophage marker, in the airways of aller- gen-exposed, RV-infected mice as well as human asthmatics experimentally infected with RV. In mice, deple- tion of macrophages decreased RV-induced eosinophilic airway inflammation and responsiveness, suggesting that these cells play a direct role in the response. Further, we found that RV-induced cytokine expression is attenuated in macrophages from TLR2 null mice, showing an unexpected role for TLR2 in the response to RV. In this proposal, we test the novel hypothesis that insertion of myristoylated viral polypeptide (VP)-4 into the macrophage plasma membrane triggers cytokine production by interacting with a TLR2 signaling complex. We have obtained abundant pilot data to support this hypothesis, including that: 1) TLR2 is required for RV- induced cytokine responses in human macrophages as well as RV1B-infected nave and allergen-treated mice; 2) transfer of IL-4 treated, wild type macrophages to TLR2 KO mice appears to be sufficient for RV-induced airway eosinophilic inflammation; 3) in macrophages, RV colocalizes with TLR2 and TLR1; and 4) recombinant myristoylated VP4 (MyrVP4) increases cytokine mRNA expression in a TLR2-dependent manner. Specific Aim 1: Determine the contribution of macrophage TLR2 and its cofactors to RV-induced airway responses. The requirement of TLR2 for RV-induced airway responses in vivo will be tested in nave and house dust mite-sensitized and -challenged mice using neutralizing antibody against TLR2, TLR2 null mice, bone marrow chimeras and a small molecule TLR2 inhibitor. The sufficiency of TLR2-expressing macro- phages will be determined by adoptive transfer. RV/TLR2 interactions will be examined by confocal microsco- py and co-immunoprecipitation. The role of putative TLR2 cofactors -- TLR1, TLR6 and CD14 -- will be exam- ined by confocal microscopy, coimmunoprecipitation and experiments employing relevant neutralizing antibod- ies and knockout mice. Finally, we will measure expression of miR-146a, a signaling outcome of TLR2 activa- tion, in nasal aspirates of asthmatic children before and during natural colds. Specific Aim 2: Determine the contribution of myristoylated VP4 to RV-induced airway responses. We will examine the direct effects of recombinant VP4, MyrVP4 and VP4 mutants on mouse and human mac- rophages in vitro and airway responses in vivo. Using fluorescence resonance energy transfer (FRET), we will examine interactions between VP4 and TLR2 and identify VP4 residues required for interaction with the TLR2 signaling complex. We will employ an RV1B maturation cleavage mutant to test if MyrVP4 is required for RV- induced cytokine expression in vitro and in vivo. Localization of VP4 and TLR2 in lipid rafts will be examined by confocal microscopy, and the requirement of TLR2 for VP4-mediated effects will be tested. Completion of this work could provide new therapeutic targets for RV-induced asthma exacerbations.